Tight epithelia, which actively transport sodium, are able to vary the rate of net transepithelial sodium transport without significantly altering their ionic content or cell volume. They do so by continually adjusting and coordinating the passive ion permeabilities of the luminal and basolateral membranes. The mechanism of this important coupling of the membranes is unknown but recent evidence suggests that cell calcium may serve this purpose. My work has centered on demonstrating an effect of calcium on the luminal membrane sodium permeability. Using a flow-quench apparatus to measure initial rate Na22 fluxes in under 100ms, I demonstrated that submicromolar calcium inhibits the luminal sodium permeability in membrane vesicles from the toad urinary bladder. The focus of my present proposal is to identify a calcium-stimulated basolateral potassium channel in the toad bladder and investigate the molecular details of the interaction between calcium and the channel. The goals of the project are: 1. Find an in vivo assay for the calcium-regulated potassium channel. My recent experiments suggest that volume regulation, following cell swelling, is the result of a calcium-dependent increase in the basolateral potassium permeability. 2. Use the cell volume-regulation assay to identify which potassium channel blockers inhibit the response and could serve as a marker for the channel. 3. Identify the K42 flux in basolateral membrane vesicles from the toad bladder which is through the calcium-stimulated potassium channel by testing the above mentioned inhibitors on the initial rate K42 fluxes measured in a flow-quench apparatus. 4. After identifying the flux in the membranes which is through the calcium-stimulated channel, test to see whether calcium acts directly to open these channels. 5. Use radiolabeled inhibitors, such as H3-phencyclidine, to identify the protein(s) within the basolateral membrane which may represent the calcium-stimulated potassium channel.